Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede

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1 Development 129, (2002) Printed in Great Britain The Company of Biologists Limited 2002 DEV Exploring the myriapod body plan: expression patterns of the ten Hox genes in a centipede Cynthia L. Hughes and Thomas C. Kaufman* Howard Hughes Medical Institute, Department of Biology, Indiana University, Bloomington, IN 47405, USA *Author for correspondence ( kaufman@bio.indiana.edu) Accepted 12 December 2001 SUMMARY The diversity of the arthropod body plan has long been a fascinating subject of study. A flurry of recent research has analyzed Hox gene expression in various arthropod groups, with hopes of gaining insight into the mechanisms that underlie their evolution. The Hox genes have been analyzed in insects, crustaceans and chelicerates. However, the expression patterns of the Hox genes have not yet been comprehensively analyzed in a myriapod. We present the expression patterns of the ten Hox genes in a centipede, Lithobius atkinsoni, and compare our results to those from studies in other arthropods. We have three major findings. First, we find that Hox gene expression is remarkably dynamic across the arthropods. The expression patterns of the Hox genes in the centipede are in many cases intermediate between those of the chelicerates and those of the insects and crustaceans, consistent with the proposed intermediate phylogenetic position of the Myriapoda. Second, we found two extra Hox genes in the centipede compared with those in Drosophila. Based on its pattern of expression, Hox3 appears to have a typical Hox-like role in the centipede, suggesting that the novel functions of the Hox3 homologs zen and bicoid were adopted somewhere in the crustacean-insect clade. In the centipede, the expression of the gene fushi tarazu suggests that it has both a Hox-like role (as in the mite), as well as a role in segmentation (as in insects). This suggests that this dramatic change in function was achieved via a multifunctional intermediate, a condition maintained in the centipede. Last, we found that Hox expression correlates with tagmatic boundaries, consistent with the theory that changes in Hox genes had a major role in evolution of the arthropod body plan. Key words: Body plan, Centipede, Chilopoda, Lithobius, Hox, labial, proboscipedia, Hox3, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal-a, Abdominal-B INTRODUCTION Over 500 million years ago in the early Cambrian, a group of animals evolved a basic morphology that would allow them to take over the world, becoming one of the most populous and diverse phyla on the planet. This group, the Arthropods, includes over a million species of spiders, mites, ticks, centipedes, millipedes, crustaceans and insects. Their segmented body plan consists of a series of repeated morphological units, which are grouped into tagmata dedicated to specific functions. Each class of arthropods has a unique division of body tagmata. For example, while the insects have three tagmata, the head, thorax and abdomen, myriapods have just two, the head and trunk (see Fig. 1). The process of tagmosis, as well as independent differentiation of individual segments, has allowed a great degree of specialization that can account for the great success of the arthropods. However, until recently, we have had little conception of the mechanism by which such body plan changes were accomplished. To understand the origin of the morphological diversity upon which natural selection acts, it is necessary to understand how the process of embryonic development evolves. We can infer the evolution of development by comparing the mechanisms of development in different species. The extensive work in Drosophila developmental genetics facilitates this, as it provides some basis for speculating about the developmental processes of other arthropods. The body plan of Drosophila is encoded in part by the patterned expression of a set of transcription factors called the Hox proteins, which divide the embryo into a series of unique domains from anterior to posterior, and thereby assign spatial identity to the segments. The Hox genes are now known to be crucial players in the development of nearly all animals, both protostomes and deuterostomes (Manak and Scott, 1994). Furthermore, because the Hox genes coordinate a large suite of downstream targets that work together to create segmental identity, a shift in the expression pattern of a Hox gene can cause major morphological change without necessarily being disastrous to the animal. Thus, changes in Hox gene expression may provide a mechanism of relatively rapid macroevolutionary change. Among the arthropods, the expression patterns of the Hox genes have been characterized in chelicerates, crustaceans and insects, with interesting implications for the evolution of the unique morphologies of those groups (see Fig. 10). Although

2 1226 C. L. Hughes and T. C. Kaufman fragments of the Hox genes have been cloned from the myriapods (centipedes and millipedes), the expression pattern of most of the Hox genes has not been determined (Cook et al., 2001; Grenier et al., 1997). As recent molecular phylogenies place the myriapods outside the insect-crustacean clade, the absence of Hox gene expression data for the group leaves a gap in the middle of the arthropod tree (Giribet et al., 2001; Hwang et al., 2001; Cook et al., 2001; Boore et al., 1998; Regier and Shultz, 1997; Friedrich and Tautz, 1995). Thus, it has been difficult to infer the full course of the evolution of these genes in the arthropods. Besides the importance of the myriapods phylogenetic position, they also have an interesting body plan. As noted, the myriapod body is divided into two tagmata, the head and trunk. The long trunk is typically fairly homonomous. That is, there is little specialization among the many pairs of legs. Moreover, the trunk can vary greatly in length and number of segments, even within a species (Minelli and Bortoletto, 1988). This relatively unspecialized, homonomous trunk is probably similar to the body plan of the arthropod ancestor. There are also interesting differences in body plan within the myriapods. The head may include two, three or four sets of mouthpart appendages (in millipedes and pauropods, symphylans, and centipedes, respectively). In centipedes, the last pair of mouthparts their notorious poison fangs is actually a modified pair of legs co-opted from the trunk and are therefore referred to here as maxillipeds. We present sequence and expression data for the Hox genes in the centipede Lithobius atkinsoni. Having established the Hox expression patterns in a myriapod, we now have data that represent all four extant classes of arthropods, and thereby are better able to infer the course of Hox evolution within this fascinating and diverse group. MATERIALS AND METHODS Centipede husbandry Wild-caught centipedes from North Carolina were supplied through Carolina Biological Supply. They were identified as Lithobius atkinsoni, thanks to help from Gerald Summers. Adult animals were housed in plastic tubs with layers of pine bark wood chips over a poured plaster-of-paris floor, with vented lids to maintain moderate humidity. Tubs were sprayed with water every few days, and crickets or mealworms were provided every few weeks. Intraspecific predation is minimal unless the animals are crowded or starved. Eggs were collected periodically by rinsing out the wood chips and tubs with water and catching the eggs in a sieve (mesh number 60). Eggs are laid year-round, and are deposited individually in damp crevices. The mother often coats each egg in a sphere of detritus; however, this is easily recognized and removed without damaging the egg. The clear eggshells allow the embryos to be staged by simple observation under a dissecting microscope. Embryos were maintained until the desired stage in watchglasses with moistened, shredded coconut fiber, which is sold through pet shops as a substrate for reptiles ( Bed-a-Beast ). Embryo preparation The extended-germband stage embryo can be seen through the eggshell at about a week after egg deposition, at room temperature. Embryos were fixed for minutes in 4% paraformaldehyde. The fixative permeates the embryo through the eggshell. After fixation, embryos were dissected from the eggshell and stored in ethanol at 20 C. Fig. 1. Arthropod body plans and phylogeny. The four major groups of extant arthropods are illustrated here, with a tree based on several recent molecular phylogenies that group the insects with the crustacea (Giribet et al., 2001; Hwang et al,. 2001; Cook et al., 2001; Boore et al., 1998; Regier and Shultz, 1997; Friedrich and Tautz, 1995). In the tree shown, myriapods are retained within the Mandibulata with insects and crustaceans (Giribet et al., 2001). Tagmatic boundaries are indicated by broken lines; names for tagmata of different groups are also indicated. Note that some groups of arthropods, for example, the crustaceans, include species with a variety of tagmatic plans not illustrated here. Cloning RNA was prepared from collections of mixed-stage embryos using Trizol reagent, following manufacturer s instructions. Total RNA was poly-a selected with the Qiagen Oligotex kit. The Boehringer Mannheim 5 /3 RACE Kit and Ambion RLM RACE kits were used to produce cdna, and PCR was performed using the Advantage2 PCR System (Clontech). Sets of degenerate primers were used to amplify portions of the various Hox genes. The primers were designed based on the sequences of orthologs from other arthropod species; primer sequences are available upon request. From the clones of the homeobox regions, exact primers were designed for 3 RACE, which produced longer clones suitable for making in situ probes. In the case of the abdominal-a gene, 3 RACE primers were designed based on the abd-a sequence of a similar centipede (Genbank Accession Number, AF362094). A variety of annealing temperatures were tested to optimize PCR amplification. A short set of five initial ramp cycles (with a gradually increasing temperature between the annealing and extension steps), or alternatively, a set of five initial touchdown cycles (with an extension temperature 5-10 C higher than the main cycles) were each found to improve amplification. The cloned Lithobius gene sequences are available through GenBank with the following Accession Numbers: labial, AF435002; proboscipedia, AF435003; Hox3, AF435001; Deformed, AF434997; Sex combs reduced, AF435004; fushi tarazu, AF435000; Antennapedia, AF434996; Ultrabithorax, AF435005; abdominal-a, AF434994; and Abdominal-B, AF Sequences of orthologs from other species used for alignments were retrieved from GenBank. The Accession Numbers are as follows: Drosophila lab, X13103; Tribolium lab, AF231104; Porcellio lab, AF148935; Lithobius forficatus lab, AF362084; Cupiennius lab, AJ007431; Drosophila pb, AAF54089; Artemia pb, AF363018; Lithobius forficatus pb2, AF362086, pb1, AF362085; Archegozetes pb, AAC35935; Drosophila bcd, P09081; Drosophila zen, P09089; z2, P09090; Tribolium zen, X97819; zen2, AF321227; Schistocerca zen, X92654; Pachymerium Hox3, CAB75744; Cupiennius Hox3, CAA06645; Drosophila Dfd, X05136; Tribolium Dfd, U81038; Thermobia Dfd, AF104005; Artemia Dfd, X70078; Pachymerium Dfd, AJ272191; Lithobius forficatus Dfd, AF362087;

3 Centipede Hox genes 1227 Cupiennius Dfd, AJ007432; Drosophila Scr, X14475; Tribolium Scr, AF227628; Artemia Scr, X70080; Ethmostigmus Scr, AF010178; Lithobius forficatus Scr1, AF362088; Scr2, AF362089; Archegozetes Scr, AF071407; Drosophila ftz, X00854; Tribolium ftz, U14732; Schistocerca ftz, X73982; Lithobius forficatus ftz, AF362090; Archegozetes ftz, AF237818; Drosophila Antp, M20705; Schistocerca Antp, U32943; Porcellio Antp, AF241662; Ethmostigmus Antp, AF010175; Lithobius forficatus Antp1, AF362091; Antp2, AF362092; Cupiennius Antp, AJ007433; Drosophila Ubx, X76210; Manduca Ubx, U63300; Artemia Ubx, X70081; Ethmostigmus Ubx, AF010179; Lithobius forficatus Ubx, AF362093; Cupiennius Ubx1, AJ007434; Ubx2, AJ007435; Junonia abd-a, L41931; Tribolium abd-a, AF017415; Artemia abd-a, X70076; Ethmostigmus abd-a, AF010174; Lithobius forficatus abd-a, AF362094; Cupiennius abd- A, AJ007436; Drosophila Abd-B, A34220; Tribolium Abd-B, AF227923; Schistocerca Abd-B, S33375; Lithobius forficatus Abd-B, AF362095; Cupiennius Abd-B, AJ Sequences were aligned using the Clustal function of MacVector software. In situ hybridization In situ probes were prepared using the Ambion MEGAscript or MAXIscript kits, with digoxigenin-utp or biotin-utp, and were mock-digested in carbonate buffer, then precipitated, resuspended and quantified. The optimal concentration of each probe was established empirically, by testing concentrations between about µg/ml. The centipede in situ hybridization protocol was developed based on multiple protocols, especially that of O Neill and Bier (O Neill and Bier, 1994), with some critical added modifications. To make the fixed embryos permeable, it was necessary to start with a 50:50 heptane/ethanol soak for 20 minutes, followed by a 1 hour soak in RIPA detergent mix [150 mm NaCl, 1% NP-40, 0.5% Sodium Deoxycholate (DOC), 0.1% SDS, 1mM EDTA, 50mM Tris-HCl, ph 8.0]. These were followed by proteinase digestion of 7.5 minutes, a post-fixation for 20 minutes, and then hybridization for up to 48 hours at 56 C. After probe was removed, a long soak of hours in hybridization buffer at 60 C helped to reduce background. Short washes in a lower-salt buffer [2 saline sodium citrate (SSC), 50% formamide, 0.1% Tween] also helped to reduce background. Antidigoxigenen and anti-biotin antibodies conjugated to alkaline phosphatase were used (Roche), with overnight incubations at 4 C. The purplish-blue stain is the result of an NBT + BCIP color reaction. (Interested readers are encouraged to contact the authors for a full, detailed in situ protocol.) Microscopy and images Developmental stages of the centipede embryos were recorded using scanning electron microscopy (Jeol). Results of in situ hybridization were analyzed and photographed through a dissecting microscope (Nikon), using a blue filter (Tiffen 80A) to correct the color balance of the halogen illumination. DAPI-stained embryos and close-ups of in situ stained embryos were photographed on a transmission microscope (Zeiss). Images were prepared using Adobe Photoshop and Illustrator, with some minor image adjustments. RESULT Embryology The extended-germband embryo of Lithobius atkinsoni is illustrated in Fig. 2. The scanning electron micrograph shows the outer form of the embryo, while the DAPI staining reveals the nuclei. The identity of each segment is labeled in the diagram. The embryo at this stage lies along the surface of the yolk, just under the chorion, with the ventral side outwards in a crescent-shape. Soon after this stage, the embryo contracts and folds in half ventrally, to form a C shape, while the dorsal membrane expands to enclose the entire yolk mass. Following this ventral flexure, the appendages elongate and differentiate, and several weeks later the hatchling emerges as a tiny centipede with eight pairs of legs. Additional leg-bearing segments are added at each molt during juvenile development, up to a final number of 15. The observed development of this species of Lithobius is consistent with that previously described for a similar species (Hertzel, 1984). Lithobius embryogenesis in general is also similar to that of other centipede families. However, the embryo is not split along the ventral midline as in the Scolopendra, as even in early stages of embryogenesis a thin layer of cells connects the left and right halves of the germband. Hox gene sequences Degenerate PCR was used to acquire short clones of homeobox regions of the genes. Using these sequences to design exact primers, we then performed 3 RACE to acquire longer clones suitable for making in situ hybridization probes. The sequences of these clones are shown in Fig. 3, aligned with homologous genes from other arthropod species. The sequences corresponding to each in situ probe are marked. Gene homology was determined by alignment with other described arthropod Hox genes from GenBank. Sequences were retrieved that corresponded to the ten Hox genes: labial, proboscipedia, Hox3/zen, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal-a and Fig. 2. The centipede extended-germband embryo is illustrated by a schematic diagram (A), a scanning electron micrograph (B) and a DAPI-stained embryo (C). Head segments are labeled in blue lettering: ocular, Oc; antennal, Ant; intercalary, Int; mandibular, Mn; maxillary I, Mx1; and maxillary II, Mx2. The labrum (Lm) probably represents the highly-modified, fused appendages of the intercalary segment (see Haas et al., 2001a; Haas et al., 2001b). The segment that will give rise to the poison fangs, or maxillipeds, is labeled in purple, as it is a trunk segment that has been co-opted into the head (Mxpd). The leg-bearing trunk segments are labeled in red (L1-L7). (The final L8 segment develops later in embryogenesis than is illustrated here.) The telson is labeled in green (Te). The stomadeum lies just behind the labrum (asterisk); the proctodeum lies to the posterior of the germband (dagger).

4 1228 C. L. Hughes and T. C. Kaufman Fig. 3. Lithobius Hox gene sequences. The partial sequences of cloned portions of the Lithobius Hox genes are aligned with orthologs from a few other arthropod species. Small arrows highlight the centipede sequences (Lithobius). Regions of the homeobox within the clones are marked above the sequences. The primers used for Lithobius are marked with boxes, indicating that that region of the sequence is somewhat uncertain. The sequence corresponding to the 5 end of each in situ probe is marked by a bar. The arrow indicates that the probe sequence extends further to the 3 end of the transcript. All sequences except those of Lithobius atkinsoni were acquired from GenBank; for Accession Numbers, see Materials and Methods.

5 Centipede Hox genes 1229 Abdominal-B. Note that although fushi tarazu and the Hox3 homologs zen, z2 and bicoid do not behave like typical Hox genes in Drosophila, they appear to have been more typical Hox genes ancestrally (see Discussion). No evidence for duplications of any of the genes was found in Lithobius atkinsoni; however, we cannot exclude the possibility of additional unrecovered Hox genes. The head genes: lab, pb, Dfd and Scr In other arthropods, the gene labial (lab) is the most anteriorly expressed of the Hox genes. Likewise, in the centipede, lab is expressed strongly in the labrum and intercalary segment, and weakly in the mandibular segment (Fig. 4A). The labrum is a thick structure that could potentially accumulate background staining as an artifact. However, staining in the labrum is seen consistently only with the lab and pb probes; therefore, we Fig. 4. The head Hox genes. (A) Two embryos stained for labial are shown, one full-length (left) and one magnified to show details of the expression pattern (right). Expression of labial is strong in the labrum (Lm) and the intercalary (Int), with weaker expression in the mandibular segment (Mn). (B) Expression of proboscipedia is shown in a younger (left) and older stage embryo (right). Staining of pb is strong in the labrum and intercalary segment, weaker in the mandibular segment and mandibular limb-buds, and strong in the maxillary I and II distal limb-buds (Mx1, Mx2). The maxillary II appendage is much longer than that of maxillary I. The arrowhead points out the expression of pb in distal maxillary II. (C) Expression of Deformed in two embryos shows expression to be across the mandibular segment, except for spots in the limb-buds (white arrow), in the segment and limb-buds of maxillary I and in a ring around the limb-bud of maxillary II (arrowhead). There is also some expression in the very posterior of the intercalary segment (black arrow). (D) Expression of Sex combs reduced is shown in a younger (left) and an older (right) embryo. In both stages, strongest expression is seen in the maxillary II segment and limb-buds, and the limb-buds only of the maxilliped segment (Mxpd). Expression near the ventral midline extends from the maxillary I to the first leg segment (arrowheads). Additional, presumptive neural expression is seen laterally in all the trunk segments (arrow). interpret this staining as a bona fide region of the expression domain for these genes. Interestingly, in both cases the labrum staining is seen in conjunction with staining in the intercalary segment. This result is consistent with a recent suggestion that the labrum represents the fused appendages of the intercalary segment (Haas et al., 2001a; Haas et al., 2001b). For the centipede embryos shown here, it should be noted that the occasional staining of the antennae is merely background accumulation. The antennae are cup-like, and in some embryos they accumulate chromagen with all probes tested, including negative control sense probes (not shown). The gene proboscipedia (pb) is expressed in very different Fig. 5. The trunk Hox genes. (A) Three embryos illustrate expression of Antennapedia. Strongest expression in is the maxilliped limb-buds and segment (arrows). Weaker expression extends to the posterior in the youngest embryo (left), but extends only from L1 to L4 in the oldest embryo on the right (bracket). The anterior boundary of expression is in the posterior of the maxillary II segment (arrowhead). (B) Expression of Ultrabithorax is shown in three embryos. From the youngest stage shown here (left) to the oldest, expression begins in the limb-buds and the posterior region (arrowhead) of the first leg segment (L1), and extends through most of the trunk. In the later stage (right), expression is absent from the last few segments of the posterior. From late extended germband stage (middle) on, expression in the trunk segments takes the form of a rosette of patches of presumptive neural tissue (arrow). (C) An early- (left) and late-stage embryo (right) show expression of abdominal-a, which is similar to that of Ubx. Expression extends from the limb-buds of L1, with a ventral boundary in the posterior of the segment (arrowheads), and extends all the way along the trunk. Expression of abd-a does not fade from the posterior in older embryos. (D) Embryos of four stages show expression of Abdominal-B. In early embryos, expression comes on in the posterior, even in cells still in the growth zone (top left), with especially strong expression circumferential to the proctodeum (bottom left; arrowhead). In extended-germband embryos, expression is strongest in the last few segments (middle), fading from L8 in the oldest embryos and then limited to the telson (right; Te). Another, weaker domain of expression is seen in segments from extendedgermband stage through older embryos (middle and right). This domain extends from the posterior of the L1 segment (arrow) on backwards through segments L2-L7 of the trunk (bracket).

6 1230 C. L. Hughes and T. C. Kaufman Fig. 6. Ubx and abd-a in early embryos. The same embryos are shown with Ubx or abd-a in situ hybridization staining (left) and with DAPIstaining (right) to facilitate identification of segments (labeled). (A) Ubx expression in a very young embryo, which has just formed the L3 segment. Expression is visible in the extreme posterior of the L1 segment (arrow), in the L2 and L3 segments, and further back in unsegmented tissue of the proliferation zone. Punctate expression is due to staining of nascent transcripts. (B) Ubx expression in an embryo that has formed five pairs of walking legs. The lateral expression is beginning to extend more anteriorly (arrowhead). (C) Expression of abd-a in an embryo that has just formed the L6 segment. The anterior boundary is at the posterior of L1 (arrow), even at the limb-bud (arrowhead). (D) Expression of abd-a in an extended-germband embryo. Now the expression domain extends into the L1 limb-buds (arrow). Abbreviations: Int, intercalary; Mn, mandibular; Mx1, maxillary I; Mx2, maxillary II; Mxpd, maxilliped; L1, first leg (etc.); PZ, proliferation zone. domains in crustaceans versus insects; thus, it was important to analyze the expression in a myriapod. The pb probes reveals a pattern of expression that extends over four segments: intercalary/labrum, mandibular, maxillary I and maxillary II (Fig. 4B). The expression is strong in the intercalary segment and labrum. In the mandibular segment, staining extends across both the segment and the limb-buds, but is weak and spotty. Expression in maxillary I and II is limited to the distal appendages. Interestingly, this expression domain resembles a combination of the crustacean and insect expression patterns (see Discussion). Expression of Deformed (Dfd) extends from the very posterior edge of the intercalary segment to the maxillary II limb-buds (Fig. 4C). Dfd is expressed across the mandibular Fig. 7. Expression of Hox3. Three embryos illustrate sequential stages of Hox3 expression. In young embryos (A,B), expression is strong throughout the mandibular limb-buds (arrowheads), with small patches of expression in part of the intercalary segment (arrows). (Staining of the antennae in A is background accumulation.) In an older embryo (C), the intercalary expression is gone, and mandibular expression is seen only in the limb-bud mesoderm (black arrowhead), and is absent from the ectodermal layer (white arrowhead). segment and limb-buds, but is excluded from the central region of the limb-buds. In maxillary I, expression extends across the entire segment and limb-buds. In the maxillary II segment, expression is only seen in the middle region of the appendages. Sex combs reduced (Scr) is expressed primarily in maxillary II and maxillipeds (Fig. 4D). In the maxillary II segment, expression is strong in the segment and the limb-buds, but in the maxillipeds expression is limited to the limb-buds. Two additional domains of expression are seen: a medial domain just outside the ventral midline, which extends from the maxillary I segment to the L1 leg segment; and, more laterally, spots of presumptive neural expression in each of the trunk segments. The trunk genes: Antp, Ubx, abd-a and Abd-B The gene Antennapedia (Antp) is expressed most strongly in the maxilliped limb-buds and segment, but is also weakly expressed in the segments and limb-buds of more posterior legs (Fig. 5A). In early stages, the posterior expression fades gradually along the entire trunk, but in later embryos, the expression reaches only to L4. The segmental expression has its anterior boundary in the extreme posterior of the maxillary II segment. Expression of the gene Ultrabithorax (Ubx) is shown for extended-germband stages of embryogenesis in Fig. 5B (expression in earlier embryos for Ubx and abd-a is shown separately in Fig. 6). In extended-germband embryos, Ubx expression is strong in the limb-buds of the first leg segment (Fig. 5B; L1), with a distinct boundary along the posterior of the L1 segment. This expression pattern of Ubx, with an

7 Centipede Hox genes 1231 Fig. 8. Expression of fushi tarazu. Embryos of eight successive stages are shown to illustrate the dynamic changes in fushi tarazu expression during development. In the younger embryos (A-D), one can see strong expression in the proliferation zone (brackets), with stripes forming in the newest segments (arrowheads). The older segments have broad expression across them, from the posterior of the maxillary I segment (arrows), across maxillary II (Mx2), on back to the posterior. In older embryos (E-H), expression has faded from the proliferation zone and from across the trunk segments, leaving the strong expression in the maxillary I and II segments (arrows; Mx2), and a presumptive neural pattern in each trunk segment (white arrows). In the oldest embryos (G,H), expression has intensified in the limbbuds of the maxilliped (arrowhead), while strong expression is maintained in the maxillary II segment (Mx2). anterior boundary in the first leg segment, is similar to that seen in a Scolopendran centipede (Grenier et al., 1997). In the early extended-germband stage, expression extends through all the segments and limb-buds of the trunk, but in later embryogenesis, expression fades from the extreme posterior. In addition, in later embryos, ventral trunk expression fades from regions of the segment, leaving rosettelike patches of expression that may be proneural. The gene abdominal-a (abd-a) is expressed in a pattern very similar to that of Ubx (Fig. 5C). In both early and late extended germband embryos, expression starts in the limb-buds and segment of L1 (again with a boundary in the posterior of the segment), and extends along the trunk. Unlike Ubx, however, the expression of abd-a does not fade away from the posterior-most segments in older embryos. Abdominal-B (Abd-B) comes on surprisingly early, in embryos still Fig. 9. Summary of centipede Hox expression. (A) The expression patterns of the ten centipede Hox genes are illustrated in cartoon form for an extended-germband embryo. Note that only the expression domains presumably corresponding to a segment identity function are illustrated here (e.g. for ftz). (B) The same expression data is shown diagramatically, for comparison of domain boundaries with each other and with tagmata and appendages of the centipede (shown for a newlyhatched larva, with seven full-size legs and an eighth not yet full length). Striped patterns indicate weaker expression.

8 1232 C. L. Hughes and T. C. Kaufman forming segments (Fig. 5D), with expression in the growth zone and a bright ring of expression around the proctodeum. In later embryos, strongest expression is seen in the last few segments, eventually becoming restricted to the telson. There is another weak domain of expression of Abd-B along the segments of the trunk, with an anterior boundary in the posterior of the first leg segment. Ubx and abd-a in early embryos The anterior boundary of Ubx and abd-a expression is presumed to play an important role in determining tagmatic boundaries in crustaceans (Averof and Patel, 1997). In addition, there has been some indication of a dynamic shift in this boundary in a centipede (Akam, 2000). Therefore, we analyzed in more detail the anterior boundary of expression of these genes in early embryos still undergoing segment formation (Fig. 6). We found that for both Ubx and abd-a, expression in early embryos is restricted slightly more towards the posterior than in older embryos. For both genes, the initial expression domain has its anterior boundary in the second leg segment (L2 in Fig. 6A-C). As the embryos age, expression becomes apparent in the posterior of the first leg segment, and eventually expression is seen in the limb-buds of the first leg segment (Fig. 6D). At none of the stages examined, from embryos with newly formed L3 segments to embryos past germband flexure, did we see expression in the maxilliped segment or limb-buds [contrary to a previous report in a similar centipede (Akam, 2000)]. Interestingly, in newly formed segments, accumulation of Ubx transcripts is low in the cytoplasm, but two distinct spots of Fig. 10. Shifting Hox domains across the arthropods. The expression domains of Hox genes from studies of various arthropods are illustrated here in simplified fashion for ease of comparison. Solid bars indicate strong expression, while striped bars indicate weak or transient expression. As this diagram represents the temporal and spatial complexity of each gene as a single bar, in some cases using information from multiple species, it is necessarily highly simplified. Therefore, we have included the source references, listed on the right (1-43), in addition to special notes on the expression patterns (a-p). For this information see below. Different arthropod species often have differing numbers of segments; the segmentboxes illustrated here are based on the spiders Cupiennius and Achaearanea (Chelicerate); the centipede Lithobius, at hatching (Myriapod); the pillbug Porcellio (Crustacean); and the firebrat Thermobia (Insect). Question marks for Hox3 and ftz indicate that these genes have not yet been analyzed in a crustacean. In the insects, Hox3 homologs and ftz have highly diverged functions, so these are treated separately in Figs 11 and 12.

9 Centipede Hox genes 1233 staining can be seen in each cell, indicative of a high level of transcription from the two chromosomal copies of the gene (not shown). At the anterior of the Ubx domain, there is a strict boundary between these Ubx-expressing cells and their neighbors that lack any detectable Ubx expression. Expression of Hox3 The Hox3 gene is expressed in the centipede in a pattern limited to the intercalary and mandibular segments (Fig. 7). Throughout embryogenesis there is strong expression in the mandibular limb-buds, with no expression within the segment. In early embryos, this domain fills the developing limb-buds (Fig. 7A), but in older embryos this domain is restricted to the interior mesodermal layer of the mandibles, with no expression in the overlying ectoderm (Fig. 7C). In addition, young embryos show expression in two small lateral patches in the anterior of the intercalary segment, just under the antennae (Fig. 7A,B). This expression is absent from older embryos (Fig. 7C). Expression of fushi tarazu The expression pattern of the centipede fushi tarazu (ftz) gene is complex, and changes dramatically throughout development (Fig. 8). In the earliest embryos expression is very strong in the proliferation zone, with stripes apparently emanating off this area (Fig. 8A,B). There is also expression in the whole of each segment up to maxillary II, with a distinct set of bands just to the posterior of the maxillary I segment (Fig. 8C,D). At subsequent stages, the proliferation zone expression becomes weaker and limited to a chevron above the proctodeum. The segmental expression gradually fades as well, except for the maxillary I and II expression, which becomes more intense (Fig. 8E). As the broad expression across the trunk segments fades, it resolves into a presumably neural pattern of small dots in a line across the anterior of each segment (Fig. 8F). In the oldest embryos, there is strong expression maintained in the maxillary II segment and a bit of the posterior of maxillary I, accompanied by expression in the limb-buds of the maxilliped segment. There is also possible weak expression in the limbbuds of more posterior trunk segments (Fig. 8G,H). Presumptive neural expression is still faintly visible in the segments of the oldest embryos examined (Fig. 8H). To summarize, ftz is expressed in the following domains: first, in the proliferation zone and the segments arising from it; then gradually stronger in the segments of maxillary I and II; later with expression in the limb-buds of the maxillipeds; and finally in the developing nervous system of the trunk. DISCUSSION The expression data for the centipede Hox genes is summarized in Fig. 9. The expression of each gene is shown References (1) Damen et al., 1998; (2) Telford and Thomas, 1998a; (3) Abzhanov et al., 1999; (4) Telford and Thomas, 1998b; (5) Damen and Tautz, 1998; (6) Telford, 2000; (7) Damen and Tautz, 1999; (8) this work; (9) Grenier et al., 1997; (10) Abzhanov and Kaufman, 1999a; (11) Abzhanov and Kaufman, 1999b; (12) Abzhanov and Kaufman, 2000b; (13) Abzhanov and Kaufman, 2000a; (14) Averof and Akam, 1995; (15) Averof and Patel, 1997; (16) Peterson et al., 1999; (17) Rogers and Kaufman, 1997; (18) Nie et al., 2001; (19) Diederich et al., 1989; (20) Rogers et al., 2002; (21) Shippy et al., 2000; (22) Pultz et al., 1988; (23) Fleig et al., 1992; (24) Brown et al., 1999; (25) Chadwick and McGinnis, 1987; (26) Kokubo et al., 1997; (27) Rogers et al., 1997; (28) Walldorf et al., 2000; (29) Curtis et al., 2001; (30) Martinez-Arias et al., 1987; (31) Wirz et al., 1986; (32) Hayward et al., 1995; (33) Zheng, 1999; (34) Nagata, 1996; (35) Kelsh et al., 1994; (36) Bennett et al., 1999; (37) White and Wilcox, 1985; (38) Tear et al., 1990; (39) Shippy et al., 1998; (40) Nagy et al., 1991; (41) Macias et al., 1990; (42) Kelsh et al., 1993; (43) Delorenzi and Bienz, Notes a Ref. 3 also reports weak staining throughout the opisthosoma. b Ref. 2 also reports staining in the opisthosoma; Ref. 3 reports two paralogs of Dfd. c In early embryos, there is also some opisthosomal staining. d Ref. 1 reports two paralogs of Ubx, and Ubx-2 mrna is expressed slightly more anteriorly than that of Ubx-1 or protein. e Additional small spots of expression in the Op2 segment correspond to the future genital pores. f Only the Hox domain of ftz is illustrated here. g In early embryos, expression of Antp extends along the entire trunk, but later fades from posterior segments. h Striped bars indicate that translation of Scr transcript in the Mx2 and T1(Mxpd) segments is delayed until late embryogenesis, where the appearance of Scr protein correlates with transformation of the maxillipeds (in Porcellio); expression is absent from Mx1 in Procambarus (Ref. 12). i Expression of Porcellio Antp is shown here; expression in Procambarus becomes restricted more to the anterior; expression in Artemia extends from posterior Mx1 to the end of the thorax (T11). j The anterior border of Ubx varies in correspondence with the number of maxilliped segments (Ref. 15); in Artemia expression extends to the end of the thorax (T11) (Ref. 14). k The top bar indicates expression of abda in Porcellio and Procambarus (although Porcellio lack the extension of expression into T7 and T8); the bottom bar indicates the expression of abd-a in Artemia. l Expression of Abd-B in Artemia is in genital segments I and II, which lie between the thorax and abdomen; the genital segments are followed by six abdominal segments that are not shown here. m The typical insect expression in the Mx and Lb segments is indicated here by a solid bar; the striped bar indicates that some insects have additional weak expression in the Mn and/or Int segments. Note that Oncopeltus lacks expression of pb in the Mx appendage, a change in expression that may be correlated to the unique sucking mouthparts of Hemipterans (Ref. 17). n The striped bar indicates that although in Drosophila expression of Scr is strong throughout the T1 segment, in other insects expression is limited to a few specific patches in the T1 segment (Ref. 27). Note that there is also expression of Scr in the mesoderm of the legs. o Expression is shown as for Thermobia; in later embryos of Drosophila expression of Antp becomes restricted to the thorax. p Expression shown is based on Thermobia and Schistocerca; in Drosophila, two Abd-B transcripts, m and r, have unique functions, and the m domain extends more anteriorly (Ref. 43).

10 1234 C. L. Hughes and T. C. Kaufman Fig. 11. Evolution of Hox3 expression and function. The expression domains of Hox3 homologs from work in other species are illustrated here in cartoon-form for comparison to that of Lithobius. In the mite, expression of Hox3 is in a typical Hox-like segmental domain, extending from the pedipalps into the opisthosoma (Telford and Thomas, 1998b). A similar Hox-like pattern is seen in spiders as well (Damen and Tautz, 1998; Abzhanov et al., 1999). Lithobius expression is also Hox-like, but is limited to the mandibular segment, plus a small anterolateral region of the intercalary segment. As indicated by the question marks, the expression of Hox3 has not yet been analyzed in a crustacean or a basal insect. Within the insects, the Hox3 ortholog zen is expressed in the extra-embryonic tissues of the grasshopper Schistocerca, the beetle Tribolium and the fruit fly Drosophila (Falciani et al., 1996; Rushlow and Levine, 1990). The extra-embryonic tissue is located primarily at the posterior pole of the Schistocerca egg, at the anterior and dorsal edge of the Tribolium egg, and along the dorsal surface of the Drosophila egg. There is also a duplicate copy of the zen gene in Drosophila called z2, which has a very similar expression pattern (Rushlow and Levine, 1990). In Drosophila, the Hox3 ortholog bicoid is maternally loaded into the anterior of the egg (Frohnhöfer and Nüsslein-Volhard, 1986). Thus, three separate functions are illustrated for homologs of Hox3 in the arthropods: a Hox-like segmental identity function (Hox3 in the mite and centipede), a function in extra-embryonic tissues (zen in the insects) and a function in early anteroposterior polarity (bicoid in Drosophila). in two diagrammatic forms. In Fig. 9A, the major expression domain of each gene is illustrated in cartoon form on an extended-germband embryo. In Fig. 9B, the extent of each gene expression domain is illustrated in bar form, below a diagram showing the segments and appendages of a larval centipede. From the intercalary segment to the telson, all segments express at least one Hox gene (Fig. 9A,B). The expression domains of the Hox genes in the centipede follow their canonical order in the complex, as known from other species (Manak and Scott, 1994). Although the genes obey this rule of co-linearity, there is a certain amount of overlap between adjacent genes. The expression of the Hox genes corresponds roughly with the tagmatic divisions in the centipede (Fig. 9B). The expression of the genes lab, pb, Hox3 and Dfd is confined to the head, while the trunk is apparently under the control of Antp, Ubx, abd-a and Abd-B. Interestingly, the maxilliped segment has expression of three genes that extend both into the head (Scr and ftz) and into the trunk (Antp). The maxilliped segment is thought to be homologous to the first trunk or thoracic segment of other mandibulate arthropods. The appendages of this segment in the centipede, however, have been highly modified. While their leg-like structure is still evident, they develop to become short and broad fangs, complete with a poison gland. Thus, the first legs of the Fig. 12. Temporal dynamics of ftz in the centipede, and evolution of ftz expression. (A) The changing expression patterns of ftz during Lithobius embryonic development are shown in cartoon form. Three major expression domains are seen: expression in the posterior, probably related to segmentation; expression in the developing nervous system; and a Hox-like expression domain in the maxillary II segment and maxillipeds. (B) The expression of ftz homologs from work in arthropod species are illustrated for comparison to Lithobius. In the mite, expression of ftz extends from the second to the fourth leg, in a typical Hox-like segmental domain (expression in the fourth limb-bud, and earlier expression in the opisthosoma are not illustrated here) (Telford, 2000). In Lithobius, both a Hox-like and a segmentation-related expression domain are seen (representations of the two expression patterns are combined onto an extended germband embryo for simplicity). In the grasshopper Schistocerca, expression is mostly in the posterior region of the growth zone, but without stripes. There are also patches of expression in the three thoracic segments, in addition to strong nervous system expression that is not illustrated here (Dawes et al., 1994). In the beetle Tribolium, expression of ftz has a pair-rule pattern, with stripes appearing out of the growth zone in alternate segments, and fading out in the anterior (Brown et al., 1994). In the fruit fly Drosophila, seven pair-rule stripes of ftz expression appear in alternating segments synchronously along the germband (Carroll and Scott, 1985).

11 Centipede Hox genes 1235 centipede are modified to become more mouthpart-like, and are used for prey capture and manipulation. This mixed head/trunk identity of the segment seems to be reflected in the Hox code found there. While the segment itself has only a trunk Hox gene (Antp), the appendages have expression of Antp as well as the head genes Scr and ftz, which are also expressed in the maxillary II segment. It remains to be determined how these genes contribute to the development of the centipede fangs. It would also be interesting to know whether the evolution of this novel appendage is correlated with a shift in the expression of these genes. Further studies of Hox expression in other myriapods such as a millipede, or functional studies in the centipede, would be very interesting regarding these issues. Shrinking domains of head Hox genes Comparing the expression of the centipede Hox genes with those of other arthropods reveals significant variability in the observed patterns (Fig. 10). For example, in the chelicerate head the Hox expression domains broadly overlap. These same genes are expressed in much more restricted domains in the head segments of crustaceans and insects. Interestingly, the expression domains of these genes in the centipede are intermediate between these two extremes. For example, the gene lab is expressed over five segments in the spider, two segments in the centipede, and only a single segment in the crustaceans and insects (see Fig. 10). Likewise, the threesegment expression domain of centipede Dfd is intermediate between the four-segment domain in the spider and mite, and the two-segment domains of the crustaceans and insects. Most striking is the comparison between expression of pb among the four groups. In the spider, pb is expressed over five segments, from the pedipalps through the fourth walking leg. In the centipede, the expression domain covers four segments, from the intercalary to the maxillary II. In the crustaceans, the expression is restricted to the antennal II segment, which is homologous to the intercalary segment. In the insects, however, the expression of pb is more posterior, limited mainly to the appendages of the maxillary and labial segments (homologous to the maxillary I and II segments of the centipede). These expression patterns suggest that the centipede may retain some Hox expression domains in an intermediate state of their evolution, from the broad domains of the chelicerates to the more-restricted, less overlapping patterns of the crustaceans and insects. Moreover, the expression domain of pb apparently became differently subdivided in different lineages; towards the anterior in the crustaceans, and towards the posterior in the insects. The centipede trunk Expression of genes along the centipede trunk is, like the morphology of the trunk, fairly homonomous. Antennapedia extends along the whole trunk in early stages, and later retracts to cover legs one through four (Fig. 5A). It is not clear whether this later, more restricted domain imparts any developmental difference to these segments, as none is evident morphologically. It is intriguing to note that this restriction to the anteriormost segments of the trunk is reminiscent of a similar restriction of Antp expression in the pleon of malacostracan crustaceans and the thorax of insects (see Fig. 10). Perhaps the domain of Antp expression was restricted to the anterior portion of the trunk in the myriapod-like mandibulate ancestor, but was only exploited fully in the specialized differentiation of the crustaceans and insects. In the centipede, Ubx and abd-a expression patterns are similarly expressed along the trunk, although Ubx expression fades from the extreme posterior segments. Expression of Abd-B is strongest in the telson, but faint expression extends over the mid-region of leg segments two to seven. As the genes Ubx, abd-a and Abd-B are likely to have similar roles in patterning the trunks of all mandibulates, we suggest that the myriapods have developed their unique body plan largely by expanding the number of segments under the control of the trunk genes. This is a similar scenario to that provided by recent findings that snakes seem to have created an elongated body by increasing the numbers of somites under the control of thoracic Hox genes (Cohn and Tickle, 1999). Genes with changing roles Those familiar with the developmental genetics of Drosophila may find it odd to refer to zen and fushi tarazu as Hox genes. In fact, only recently have these been recognized as such. Yet recent studies indicate that these genes were probably typical Hox genes in the arthropod ancestor, but have undergone remarkable functional transitions in some arthropod lineages. The expression of the insect orthologs of Hox3 bicoid, zen, and z2 reflects a remarkably versatile repertoire of functions (see Fig. 11). The gene bicoid encodes an anterior-specifying morphogen deposited maternally into the Drosophila egg (Frohnhöfer and Nüsslein-Volhard, 1986). The gene zerknüllt (zen) plays a role in the specification of Drosophila extraembryonic tissues, whereas z2, the adjacent duplication of zen, has a similar expression pattern but no discernable function (Pultz et al., 1988; Rushlow and Levine, 1990). When homologs were cloned from other insects, it was realized that these genes had sequence similarity with both the Hox3 genes of vertebrates as well as the zen gene of Drosophila; thus, zen is actually a highly derived homolog of Hox3 with a novel function (Falciani et al., 1996). More interesting still, when bicoid and zen homologs were cloned from another fly, it was discovered that these genes have sequence similarity as well (Stauber et al., 1999). Therefore it is likely that, despite its allimportant role in early Drosophila development, bicoid may actually be a fairly recent duplication of the zen gene that has diverged greatly in function. Thus, the Hox3 gene has apparently been caught in the act of changing function drastically in evolution twice! To those interested in understanding the mechanisms of gene evolution, such a gene is worthy of much study. Researchers are currently working to clarify the timing of the zen to zen + bicoid duplication and divergence in the higher insects (Stauber et al., 1999; Stauber et al., 2000). The results we present are relevant to the earlier functional change, from Hox3 (with a Hox-like role) to zen (with a role in extra-embryonic tissues). In spiders and a mite, the Hox3 gene has a typical Hox-like expression pattern, with a broad domain whose anterior boundary is approximately co-linear with the other Hox genes (Telford and Thomas, 1998b; Damen and Tautz, 1998; Abzhanov et al., 1999). The homologous genes of the grasshopper Schistocerca and the beetle Tribolium apparently have zen-like roles, with expression in the extraembryonic serosa (Falciani et al., 1996). The centipede Hox3